Advances in the control of malaria are threatened by the spread of drug-resistant parasites. There is a great need for new antimalarial chemotherapy and more fundamentally, for new chemotherapeutic targets. We have discovered that the active principle in microbial preparation of the aspartic protease inhibitor pepstatin is an esterified derivative that acts as a prodrug. Pepstatin esters show nanomolar potency against Plasmodium falciparum in culture, with an IC50 for the hexyl ester of 19 nM. This suggests that the target of pepstatin esters, though yet to be elucidated, is druggable. To identify the cellular target of these compounds we have, with difficulty, selected resistant mutant parasites. The mutants have alterations in a small protein of unknown function that we call TITLE. We propose to investigate whether or not TITLE is the direct target of pepstatin esters. We will obtain geneti evidence for the role of TITLE in resistance to pepstatin esters, will perform chemical biology studies to identify the direct target and will find interacting proteins for TITLE and for the diret target if that is different from TITLE. Specifically: 1) We will perform allelic replacement studie to establish that TITLE is important for resistance. TITLE could be acting as the PSE target, as a modifier of the target or as a modifier of the compound. We will perform overexpression studies using wild-type and mutant TITLE alleles to gain insight into TITLE's role. We will assess PSE accumulation and modification in wild-type and mutant parasites. 2) We will perform pull-down studies using a biotinylated, photo-cross linkable version of pepstatin. This will enable us to confirm cellular interaction of TITLE with inhibitor, or to identify the real target if TITLE isnot the direct binding protein. We will take advantage of differential binding of esterified vs. non-esterified pepstatin to ensure specificity. 3) We will tag and pull down TITLE using anti-tag and anti-TITLE antibodies. If TITLE is not the direct target, the target identified in aim 2 will be similarly tagged and pulled down. The target will also be pulled down with derivatized pepstatin as above but washed less stringently to preserve interactions with other proteins. We will perform MS-MS analysis on the pull-downs and will confirm interacting proteins by reciprocal immunoprecipitation. The experimental plan comprises a novel parallel pull-down approach. We anticipate that these studies will allow us to establish the target of PSEs, will yield insights ino PSE mechanism and will provide us with a promising new avenue for antimalarial drug development.